Automatic analyzer

ABSTRACT

An amino acid analyzer having an ion exchange column through which buffer reagents are passed is provided with circuitry means for automatically effecting the various steps of the analysis. The circuitry means being effective to allow continuous automatic supply of samples to the ion exchange column and also to automatically control the elution process, permits uninterrupted continuous operation of the analyzer without the necessity of operator attention. A central timer mechanism effects actuation of a sample supply system to place the sample on the ion exchange column and thereafter the central timer mechanism sequentially activates other timers to automatically control the eluting process. During the eluting process the analysis is carried out in the conventional manner, i.e., using a ninhydrin system and a colorimeter. The colorimeter detects the concentration of amino acids present in the column effluent and sends electrical signals to a recorder mechanism to record or plot the quantity of specific amino acids present in the sample.

United States Patent Schneider Mar. 14, 1972 [54] AUTOMATIC ANALYZERPrimary Examiner-Morris O. Wolk [72] Inventor. Eugene Louis SchneIder,St. Louis, Mo. Assistant Examinelgk' E Serwin [73] Assignee: RalstonPurina Company, St. Louis, Mo. Attorney-R Bl'ukaldt and Hurst [22]Filed: NOV. 22, 1968 57 ABSTRACT [21] App]. No.: 778,048 An amino acidanalyzer having an ion exchange column through which buffer reagents arepassed is provided with cir- 52 US. (:1. ..23 2s3 R, 23/230 A, 23/253 A,witty means P effeFting theyarious Steps of 210/31, 210/198, 210/200,73/61 1 C the analysis. The circuitry means being effectiveto allow con-[51 im. Cl ..G01n 31/04, GOln 31/08 autommlc supply Samples exchange 531Field of Search ..23 253, 253 A, 259, 230 A, aummatically OM01 F Pmcess,23/230; 73 /61 1 C; 2l0/31 198 200 permits uninterrupted continuousoperation of the analyzer without the necessity of operator attention. Acentral timer [56] References Cited mechanism effects actuation of asample supply system to place the sample on the ion exchange column andthereafter UNITED STATES PATENTS the central timer mechanismsequentially activates other timers to automatically control the elutingprocess. During the 3,230,048 H1966 Skeggsm ..23/253 eluting process theanalysis is carried out in the conventional 3,266,322 8/1966 Negersmnhet X manner, i.e., using a ninhydrin system and a colorimeter; The3'334969 8/1967 Catravas "23/253 X calorimeter detects the concentrationof amino acids present 3,341,299 9/1967 Catravas "23/253 X in the columneffluent and sends electrical signals to a gase at recorder mechanism torecord or plot the quantity of specific r ma l 3373 872 3/1968 Hrdina..23/253 UX ammo aclds m the Sam 6 3,518,874 7/ 1970 Hrdina ..23/253 R12 Claims, 4 Drawing Figures PATENTEDMAR 14 1972 3. e49 ,203

SHEET 1 0F 3 9 8 Illllllllllllll Q -2 12e 1 2d 12c 12b 6 t1 t1 (:1 b 1INVENTOR EUGENE LOUIS SCHNEIDER BY ATTORNEY FIG. 3

AUTOMATIC ANALYZER BACKGROUND OF THE INVENTION Amino acid analyzerswhich utilize the principle of ion exchange have been fairly common inthe instrumentation field. These analyzers make use of the principle ofdepositing a sample to be analyzed on the resin of an ion exchangecolumn and thereafter pass various buffer reagents successively throughthe column, a ninhydrin reaction and a colorimeter to obtain data whichreflects the concentration of the various amino acid constituentspresent in the sample being analyzed.

In the prior art, it has been necessary for an operator to present eachsample which is to be analyzed to the machine by manually inserting thissample on top of the ion exchange column. Not only was this undesirabledue to the necessity of an operator manually placing each sample to beanalyzed into the machine but also it was necessary that the seals onthe ion exchange column be opened to permit the insertion of suchsamples. The necessity of continuously opening these seals resulted inthe attendant problems of seal leakage due to the high pressures of thefluids used in the ion exchange columns.

Other prior art attempts to automate the amino acid analysis system haveutilized an automatic method of inserting a cartridge containing thesample onto the end of the ion exchange column. Although this methodeliminates some of the necessary operator attention, it still requiresthe periodic opening of the seal on the column to receive samplesthereby permitting leakage of the fluids around the seal. In addition,the samples were mounted in such a manner that the heat generated by theanalyzer in its operation was effective to destroy some of the aminoacid constituents, such as glutamine and methionine, in the sample priorto its analysis.

In an effort to overcome these aforementioned undesirable features,applicant has developed a system which utilizes time controlledcircuitry means for actuating the various components of the analyzerenabling the operation of such an analyzer to be completely automaticwithout the necessity of operator attention.

Another object of the present invention is to provide an analyzer whichpermits samples to be maintained separate from the column portion of theanalyzer. These samples may even be maintained under refrigeratedconditions prior to their analysis, thereby minimizing the possibledeterioration of the various amino acid constituents.

Another object of the present invention is to provide circuitry meanseffective for actuating a sample feed mechanism which delivers thesample to a control valve and in response to other electrical impulsesfrom said circuitry means said control valve is effective to supply thesample to the ion exchange column and control the eluting process.

Another object of the present invention is to provide an automatic aminoacid analyzer which permits sequential feeding of samples to be analyzedto the apparatus without the necessity of opening the high-pressureseals on an ion exchange column.

Another object of the present invention is to provide an amino acidanalyzer which is provided with a central timer mechanism tosequentially effect the analysis of a sample and said timer mechanismbeing responsive to the completion of the analysis to automaticallysupply another sample to be analyzed.

Another object of the present invention is to provide circuitry means onthe amino acid analyzer which permits automatic operation and whichpermits the columns to be run in overlap condition. It should beunderstood that overlap condition means that when two columns are usedfor the analysis, the eluting process may be initiated on the longcolumn prior to the completion of the eluting process on the shortcolumn. The long column may be started prior to completion of the shortcolumn due to the existence of an initial period of operation on thelong column when there are no amino acids of interest which are eluted.

Still another object of the present invention is to provide automaticcircuitry means which permits the short column eluting process to beginas the long column is being stripped and prepared for another sample.

These and other objects and advantages of the present invention willbecome more apparent in the description hereinafter disclosed.

Briefly, the present invention comprises an automatic amino acidanalyzer having an ion exchange column, a control valve for controllingthe supply of samples to the column and for controlling the elutingprocess, sample supply means for supplying a predetermined amount ofsample to the control valve, and a central timer means for controllingthe analysis cycle which includes energizing said sample supply means tosupply the sample to said control valve, said central timer meansactivating said control valve to initially supply the sample to the ionexchange column and thereafter controlling the eluting process, and saidcentral timer means being responsive to the completion of the elutingprocess to begin the analysis cycle for another sample.

DESCRIPTION OF THE DRAWINGS FIG. 1 is a view of the amino acid analyzerembodying the present invention,

FIG 2 is a diagrammatic view of the fluid pressure system of the aminoacid analyzer of FIG 1,

FIG 3 is an enlarged diagrammatic view of the flow path through one ofthe control valves of the amino acid analyzer, and

FIG 4 is a schematic wiring diagram of a typical electrical circuit forthe present amino acid analyzer.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring now to FIG 1, anamino acid analyzer or chromotography apparatus 1 is shown as having acabinet 2 which is provided with a refrigeration portion 3. Therefrigeration unit 3 houses or contains a turntable 4 adapted to receivesamples 5, for subsequent analysis by the apparatus 1. Ion exchangeresin columns 6 and 7 are provided on the cabinet 2 with the resinlength of column 6 being approximately 7 cm. while the resin length ofcolumn 7 is approximately 51 cm. Solenoid operated control valve means 8and 9 are provided on the cabinet 2 for controlling the flow of samplesand buffer reagents to the columns 6 and 7, respectively. Sample feedmechanism or dipper 10 is provided in the refrigeration unit 3 forselectively supplying samples 5 to the control valves 8 and 9, and thesamples 5 are subsequently deposited or absorbed on the resin of columns6 and 7. A central time control mechanism 11 is affixed to the cabinet 2for automatically effecting movement of the various components of theamino acid analyzer 1 to automatically complete an analysis of a samplein a predetermined time sequence, as will be described in detailhereinafter. A series of individual timers 12a, 12b, 12c, 12d, 12c and12f are provided for controlling specific portions of the amino acidanalysis as it is carried out in an automatic manner by the apparatus 1.A recorder 13 is provided for recording curves or peaks which reflectthe concentrations of the various amino acids present in the samples asthey are separated or eluted from the resin columns in response to thepassage of buffers therethrough. It should be understood that as analternative, a computer or an integrator, such as available fromInfotronics Company model CRS-l in, may be utilized as a digital readoutsystem or that both a computer and recorder 13 may be utilized tocompile the results of the analy- SIS.

Referring now to FIG 2, a diagrammatic view of the fluid pressure systemof the analyzer is shown with the flow lines in the control valves 8 and9 illustrating their activated positions to receive a portion of asample 5 from the turntable 4. The sample feed mechanism or dipper 10(shown here in its activated position) is controlled by way of a motor14 and gears 15 to effect its movement between an upright or inactivatedposition and an activated position wherein the dipper is in the on theturntable 4. The dipper 10 is connected by means of teflon tubing 16 tothe inlet port 17 of the eight-port solenoid operated control valve 9.Bypass ports 18 and 19 of the control valve 9 are connected to eachother by means of teflon tubing 20 and the sample ports 21, 22 of saidvalve are connected by the teflon tubing 23. The tubing 23 has therein aseries of sample retention loops 24 arranged so that a sample ofconvenient size, such as 1 millileter, may be held in such loops. Thebuffer inlet port 25 is connected by means of tubing 26, a pump 27 andtubing 28 to a solenoid operated selection valve 29. The selection valve29 is connected by means of tubing 30a, 30b, 30c and 30d to the bufferreagent supplies 31, 32, 33 and 34.

The buffer reagents 31, 32 are selected to carry out the completeeluting process on the column 7. The buffers 33 and 34 are selected toregenerate the resin of said column with the buffer 33 serving to stripthe residue of the sample from the column and the buffer 34reconditioning the resin in said column. In this particular apparatus,it has been found desirable to utilize the buffer 31 as a solutionhaving a pH of approximately 3.25, and the buffer reagent 32 as asolution having a pH of approximately 4.1 l. The buffer reagent 33 isNaOH and the buffer 34 is equilibration, which are selected tocompletely strip the sample from the resin of column 7 and prepare itfor receiving another sample. The selection of these buffers may dependon the operator's preference.

The outlet port 35 of the control valve 9 is connected by teflon tubing36 to the sample inlet port 37 of the eight-port solenoid operatedcontrol valve 8, Another outlet port 38 of the control valve 9 isconnected by tubing 39 to the inlet port of the long ion exchange column7. The outlet port of the long column 7 is connected by teflon tubing 40to a solenoid operated valve 41 which may be operated to divert thefluid supplied thereto to the the drain line 42 or to the teflon tubing43 which is connected to a mixing valve 44.

Bypass ports 45 and 46 of the control valve 8 are connected to eachother by means of teflon tubing 47 and the sample ports 48, 49 of saidvalve are connected by the teflon tubing 50. The tubing 50 has therein aseries of sample retention loops 51 arranged so that a sample ofconvenient size, such as l millileter, may be held in such loops. Thebuffer inlet port 52 is connected by means of teflon tubing 53, a pump54 and tubing 55 to the valve 56. The valve 56 is connected by means oftubing 57 to buffer supply 58. The buffer is a solution having a pH ofapproximately 5.18, however, this may be varied depending upon theoperator's preference. The outlet port 59 of the control valve 8 isconnected by tubing 60 to the sample supply pump 61 and said pump isconnected to a drain 62. Another outlet port 63 of the control valve 8is connected by tubing 64 to the inlet port of the short ion exchangecolumn 6. The outlet port of the short column 6 is connected by tubing65 to a valve 66 which may be operated to divert the fluid suppliedthereto to the drain line 67 or to the tubing 68 which is connected tothe mixing valve 44. A ninhydrin system 69, which is well known in theart, is connected by means of tubing 70 to the mixing valve 44 andtubing 71 connects the mixing valve 44 with a standard reaction bath 72.Tubing 73 is connected to the reaction bath 72 and passes through acolorimeter 74 and therefrom to a drain.

Referring now to FIG 3, the flow diagram for the control valve 9 isshown. Since the control valve 9 and control valve 8 are identical onlythe flow pattern through the control valve 9 will be discussed indetail, it being understood that the flow through the control valve 8will be identical. The control valve 9 is a commercially availableeight-port valve having a plunger 9a therein which control the pressurefluid flow through the valve. The plunger 9a is spring biased to itsnormal or deactivated position establishing pressure fluid flow throughthe valve 9 as shown by the solid lines of FIG 3 whereby the sample maybe deposited on the ion exchange resin column 7 and whereby the bufferssupplied through the tubing 26 may be passed through said column. Theplunger 9a is movable in response to fluid pressure from a source (notshown). The fluid pressure is applied to one end of the plunger 90 by asolenoid valve to move said plunger to a position establishing pressurefluid flow through the valve as shown by the dotted lines whereby thesample may be supplied from the tubing 16 to the sample retention loop24. More particularly, when solenoid controlling the control valve 9 isdeactivated the plunger 90 is in its normal or deactivated position, andthe bufi'er supplied to the column 7 will pass through tubing 26, sampleloop 24 and therefrom through the port 38, tubing 39 and into the column7. When the solenoid is activated, the plunger mechanism 9a is activatedor moved from its eluting position. The fluid flow between the ports 25,22 and ports 21, 38 is interrupted and pressure fluid communication isestablished between the ports 17, 35 through the loop 24 and the ports25, 38 through the bypass tubing 20. This enables the sample to bedelivered from the tubing 16 to the sample retention loop 24.

Referring now to FIG 4, a simplified schematic drawing of the typicalelectrical circuits to be used with the apparatus of FIG 1 is shown. Thecircuits primarily consist ofa long column timing circuit indicatedgenerally at 75, a short column timing circuit indicated generally at 76and the remaining portion of the circuitry controlled by the centraltimer mechanism 11. Central timer mechanism 11 consists of a standardindustrial sequential l0 terminal timer which is commercially availablefrom a supplier such as Mallory & Company. A manual start switch 77 isprovided between a power source (not shown) and terminal 780 of thecentral timer 1 l.

The terminal 78b is connected to another terminal 79b on the centraltimer l1 and to the motor 80 of said timer. It should be noted that theswitches 78, 79 connecting the terminals 78a, 78b and 79a, 79b are soprovided that one or the other of them is closed at anytime. Therefore,when the terminals 78a, 78b are connected, the terminals 79a, 79b areopen and when 78a, 78b are open, the terminals 79a, 79b are closed, Theswitch 81 on the timer 11 is connected to the power source and to themotor 14, said motor being effective through means of the gears 15 toraise and lower the dipper mechanism 10.

Another switch 82 on the timer 11 is connected between the power sourceand the motor 83 which is effective in rotating the turntable 4 topresent a new sample 5 to the dipper mechanism 10. The switches 84, 85and 86 on the timer 1 1 are connected between the power source and themotor of the sample supply pump 61, and the solenoids 87, 88respectively. The solenoids 87, 88 are effective in controlling theactuation of the plungers ofthe control valves 8 and 9, respectively.

The short column timer circuit 76 has a standard commercially availabletimer 12a therein, available as model 3053 from such suppliers as ATC.The timer 12a is connected to a power source. Also, the timer 12a isprovided with the normal means of manually setting the time period whichwill elapse before the contacts therein are closed and the timer timesout. The timer 12a is connected to switch 90 in the timer l1 and amanual push button switch 91 may also be provided between the timers 1 land 12a. The terminal 92 of the timer 12a is connected to the motor ofthe buffer pump 54, the solenoid 93 of the valve 41 and to the terminals94, 95 which serve as the power source for the pump of the ninhydrinsystem 69 and the recording mechanism 13. Another terminal 96 of thetimer 12a is connected to the terminal 790 of the timer l1 and a switch97 is provided intermediate the two timers.

The long column timer circuit 75 is provided with a series of individualtimers 12b, 12c, 12d, 12c and 12f. All of these timers are provided withthe normal means of manually setting the time period which will elapsebefore the contacts therein are closed and the timer times out. Each ofthe timers 12b, 12c, 12d and 12e are effective in controlling the lengthof time that the buffers 31, 32, 33 and 34 are permitted to flow throughthe long column 7. The timer 12b is connected to a power source and tothe switch 98 on the central timer 11. A manual pushbutton switch 99 maybe provided between the timers 11 an 12b, The terminal 100 on the timer12b is connected to the motor of the buffer supply pump 27 and to thetimer 12f. Terminal 101 of timer 12b is connected to the solenoid 102 ofthe selection valve 29 and to the timer 120. Also, terminal 103 on timer12b is connected to terminal of timer 12c.

The terminal 120 is connected to a solenoid 105 of the selection valve29 and to the timer 12d. Timer 12d is connected to the solenoid 106 ofthe selection valve 29 and the timer 12e. It should be noted that thetimer 12e and 12f are connected to a power source, Also, it should benoted that terminal 107 of the timer 12e is connected to the switch 97which in turn is connected to the terminal 79a.

The terminal 108 of the timer 12f is connected to the solenoid 109 ofthe valve 66 and to the terminals 110, 111 which serve as the powersource for the pump of the ninhydrin system 69 and the recordingmechanism 13. The terminal 112 of the timer 12f is connected to theterminal 780 of the central timer 11 and a switch 113 is providedintermediate to these two timers.

The switch 113 is maintained in a closed position when a sample 5 ispresented beneath the dipper mechanism 10 by means of a springarrangement 114 and an actuation arm or sample detector 115 which isprovided in a collar 116 on the machine 1. When the actuation arm orsample detector 115 is in contact with the sample 5 in the turntable 4the switch 113 is closed, however, if a sample 5 is not present beneaththe dipper 10 the spring 114 will force the sample detector rightwardlyto open the switch 113.

Samples 5 for the amino acid analyzer are prepared by the standardhydrolysized process, e.g., a 125 ml. sample is mixed with six normalHCl, purged with nitrogen, sealed and heated at 110 C. for approximately22 hours, An internal standard may be added which is a natural aminoacid and which will be eluted as a separate peak. To complete the sample5, an aliquot sample is taken, dried, and thereafter a buffer having apH of 2.2 is added to obtain a 10 ml. sample. The samples are thenplaced in cups on the rotary turntable 4. Since the turntable 4 islocated in the refrigeration portion 3, a large number of samples 5 maybe placed therein without concern that some of the amino acidconstituents of the samples will be destroyed.

In the operation of the amino acid analyzer 1 the control valves 8 and 9are normally in a position as shown in FIG 3, that is, permitting fluidflow from the buffer system to the ion exchange columns 6 and 7, throughthe sample retention loops 24, 51. Also, the heating unit of thereaction bath 72 and the colorimeter 74 are maintained energized; theturning on of the recorder 13 being effective to receive the signalsfrom the colorimeter. In response to the closing of the manual switch 77current from the power source flows through the switch 78 to energizethe timer motor 80. After the elapse of a predetermined amount of time,the switches 84, 85 and 86 are closed thereby serving to energize thepump 61 and activate the control valves 8 and 9. The control valves 8and 9 are then in a position to establish pressure fluid communicationtherethrough as shown by the lines in FIG. 2. After the elapse ofanother predetermined amount of time, the switch 82 is closed serving toactivate the motor 83 and rotate the turntable 4. The switch 82 isopened after a predetermined time has elapsed which is sufficient topermit the turntable 4 to rotate an amount to properly locate a sample 5below the dipper mechanism 10. It should be understood that since thesample supply pump 61 has been activated while the dipper 10 is not inthe sample 5, air is passed through the supply line and sample loops 24,51 to serve as a flushing or cleansing action prior to insertion of thesample into said loops.

Thereafter, the timer 1] closes the switch 90 thereby serving toenergize the circuit 76 and activate the short column timer 120. Thisserves to connect the power source of the timer 12a to the terminal 92.When the power source of timer 12a is connected to terminal 92 thebuffer pump 54 is activated serving to force the buffer through theshort column 6 to develop the desired fluid pressure in said columnprior to injection of the sample. The timer 12a is also effective inenergizing the solenoid 93 to activate the control valve 41 to establishpressure fluid communication between the conduits 40 and 43.Simultaneously, the power to the terminals 94, 95 serves to activate thepump of the ninhydrin system 69 and to activate the recorder 13. Oncethe timer 12a has been activated the switch 90 in the central timer maythen be opened and the timer 12a will operate for the period of time seton said timer. The timer mechanism 11 then closes the switch 81 whichserves to insert the dipper 10 into the sample 5 and since the samplesupply pump 61 is already activated the sample is drawn into the sampleloops 24 and 51 of the control valves 9 and 8, respectively. After aperiod of time which is sufficient for the sample 5 to be pumped intothe control sample loops 24 and 51, as previously described, the timer11 opens the switch 84 which serves to turn off the sample supply pump61.

Thereafter the switch 86 is opened to deactivate the solenoid 88 whichserves to move the plunger of the control valve 8 to its normal ordeactivated position establishing pressure fluid flow through saidcontrol valve as shown by the solid lines of FIG 3. Also, the switch 78is opened while the switch 79 is closed which serves to interrupt thepower source to the motor 80, thereby turning off the timer mechanism11. The manual switch 77 may then be opened and thereafter the analyzer1 will operate in a completely automatic manner as describedhereinafter. The total elapsed time from the start until the manualswitch may be opened is approximately 8 minutes.

The buffer 58 is pumped through the sample loop 51 and into the column 6which permits the sample contained in said loop to be absorbed on theresin of said column. Thereafter as the buffer 58 passes through thecolumn 6 the eluting process is performed wherein the effluent from saidcolumn passes through the mixing valve 44 to be admixed with theninhydrin and therefrom through the reaction bath 72 where the effluentis heated and therefrom through the colorimeter 74. The colorimeter 74is effective to transmit electrical signals, in response to theconcentration of amino acids present, to the recorder 13 to form peaksor curves, in the conventional manner. The eluting operation on theshort column 6 is effective to produce peaks which typically are l) atotal of the amino acids other than the bases, (2) lysine, (3)histidine, (4) ammonia, and (5) arginine. This eluting process on theshort column 6 takes approximately minutes, however, it should beunderstood that the timer 12a may be set for any period of time desired.Upon completion of the time cycle of the timer 12a, the power source isdisconnected from terminal 92 and connected with terminal 96. Thisconnection will be maintained until another impulse is received by thetimer 12a through the switch or pushbutton 91. This serves to turn offthe pump 54 and to deactivate the solenoid 93 which permits the valve 41to connect the tubing 40 with the drain line 42. In addition, the powersupply to the terminals 94 and 95 is interrupted to turn off the pump ofthe ninhydrin system 69 and the recorder 13. It should be understoodthat since the buffer 58 is effective to remove all of the sample fromthe resin of column 6 it is not necessary to pass further reagentsthrough the column before another sample is presented thereto.

The connection of the power source to the terminal 96 of the timer 12ais effective through the switch 79 to transmit an impulse from the powersource to the motor 80 and again energize the timer mechanism 11.Thereafter, the switch 98 is closed which transmits an impulse to thetimer 12b and actuates the long column timer circuit 75, as will bediscussed in detail hereinafter.

It should be understood that the power source of timer 122 is normallyconnected with terminal 104 and therefore with terminal 103 on timer12b. Thus, when the timer 12b is turned on, the terminal 103 isconnected with terminal 100 which serves to turn on the motor of thebuffer pump 27. The activation of the buffer pump 27 serves to force thebuffer 31 through the long column 7 to develop the desired fluidpressure in said column prior to injection of the sample. The energizingof the timer 12b is also effective to transmit an electrical impulse tothe timer 12f which turns on the timer 12f and connects its power sourcewith the terminal 108. The timer 12f is effective in energizing thesolenoid 109 to activate the control valve 66 to establish pressurefluid communication between the tubing 65 and 68. Simultaneously, thepower to the terminals 1 10 and 11 1 serves as the power source toactivate the pump of the ninhydrin system 69 and to activate therecorder 13 for receiving signals from the colorimeter 74. Once thetimer 12b has been activated the switch 98 in the central timer may beopened. Thereafter, the switch 85 is opened to deactivate the solenoid87 which serves to move the plunger of the control valve 9 to its normalposition establishing pressure fluid flow through the sample loop 24 ofsaid control valve as shown by the solid lines of FIG 3. The timer 11then opens the switch 79 while the switch 78 is closed which serves tointerrupt the power source to the motor 80, thereby turning off thetimer mechanism 11.

With the control valve 9 moved to its normal position, the buffer 31 ispumped through the sample loop and into the column 7 which permits thesample contained in said sample retention loop to be absorbed on theresin of said column. Thereafter, the eluting process is carried out bythe buffer 31 for the period of time set on the timer 12b. The buffer 31passes through the column 7 and then follows the normal course throughthe mixing valve 44 where ninhydrin is added and therefrom through thereaction bath 72 and colorimeter 74. When the period of time set on thetimer 12b has elapsed the eluting process to be carried out by thebuffer 31 is completed. in a typical operation of the analyzer 1, thetimer 12b is set for approximately 95 minutes, but this may be varieddepending on the operators preference. The timer 12b then connects theterminal 103 with the terminal 101 while maintaining the connection ofterminal 103 with the terminal 100. The connection of terminal 101 withthe power source serves to supply an electrical impulse to the timer 12cthereby beginning the timing cycle controlled by this timer. The impulsewhich activates the timer 120 also activates the solenoid 102 whichmoves the selection valve 29 to a position establishing pressure fluidflow of the buffer 32 through the ion exchange column 7 and theremainder of the analysis system. Upon completion of the final time seton the timer 120, amino acids which were not separately eluted duringthe short column run will have been recovered. In a typical operation ofthe analyzer l, the timer 12c is set for approximately 60 minutes,however, this may be varied by the operator. Therefore, timer 12b and12c control the complete eluting cycle to recover the amino acids notpreviously separately recovered on the short column 6. In addition, byincluding an internal standard in the sample as previously discussed,the curve produced in the recovery of this standard may be compared witha known curve so that the true ratio of recovery from the sample 5 willbe known.

Upon completion of the timing cycle of timer 12c, an impulse is sent tothe timer 12d and to the solenoid 105. The solenoid 105 being effectiveto move the selection valve 29 to a position permitting the bufferreagent 33 to be passed through the ion exchange column 7.

Since the buffer 33 is NaOH the passage of this buffer through thecolumn 7 serves to completely strip the remaining sample present on theresin of said column. it should be understood that the materialremaining on the resin column which is stripped by the buffer 33 is thesame amino acid constituents which were previously eluted from thecolumn 6. Upon completion of the timing cycle of the timer 12d the ionexchange column is completely stripped of all of the sample 5 depositedthereon. An impulse is sent from the timer 12a to the timer l2e and alsoto the solenoid 106 which moves the selection valve to a positionpermitting pressure fluid flow of the buffer 34 through the column 7.The passage of the buffer 34 through the column 7 serves to reconditionthe resin in said column so it will be receptive and have a high degreeof affinity for the introduction of another sample to said column. Thus,the timers 12c and 12d control the complete regeneration operation oncolumn 7. In a typical operation each of the timers 12c and 12d are setfor 35 minutes but this may be varied. The eluting operation and theregeneration operation are both controlled by the long column timingcircuit 75. Upon completion of the timing cycle [22, the power source tothe terminal 104 and the timer 12b is interrupted, serving to effect adeenergization of the buffer pump 27. This also permits each of thetimers 12b, 12c, 12d and 12e to return to their original positions andtime settings.

In addition, it should be understood that the timer 12f may be set totime out or elapse simultaneously with the timing out of the timer 12e.The timing out of the timer 12f serves to interrupt the power supply tothe terminal 108 thereby deactivating the equipment connected theretoand also serves to connect the terminal 112 to the power source of saidtimer. With the terminal 112 connected to the power source an impulse issent to the switch 78 of the timer mechanism 11 and thereafter anotheranalysis cycle as previously described is begun. The power source willbe maintained in contact with the terminal 112 until another impulse isreceived by the timer 12f from the terminal 100. It should be noted thatwith the sample detection switch 113 in the line connecting the terminal112 to the switch 78 the absence of a sample 5 on the turntable 4permitting the switch 113 to open will prevent a further power supply tothe motor and shut down the analyzer 1. However, if another sample 5 ispresent, the analyzer 1 will continue to operate in the analysis cycleas previously described.

Further, it should be understood that by adjusting the timer 12f suchthat it expires upon completion of the complete eluting operation orsimultaneously with the completion of the eluting operation controlledby the timer 12c, it is possible to begin another analysis cycle whilethe timers 12d and 122 carry out the regeneration operation of strippingand reconditioning on the long column 7. This is achieved due to theability of the control valve 9 to permit the buffer reagents 33 and 34to be cycled into the column 7 through the bypass 20, while a sample issimultaneously being pumped into the loop 24 as previously described.Thus upon completion of the eluting operation of timer 120, the timer12f would time out deactivating the equipment connected to timer 12f andconnecting terminal 112 with terminal 78 as described heretofore. Sincepump 27 is connected to timer 12b, it will not be deenergized until thetimer 12c has timed out. In this manner, it is possible to supplyanother sample to the control valves 8, 9 and begin analysis of thesample on the short column 6 while the regeneration steps are carriedout of the column 7.

It should also be understood that when the switch 97 is in the positionshown in FIG 4 the apparatus is in a position to operate in its normalmanner. By this is meant that upon completion of the short columnanalysis a signal is sent through the switch 97 to the timer 1] andthereafter the long column analysis is begun. By moving the switch 97 toits lower position to connect the terminal 107 of timer 12 with thetimer 79a, the analyzer 1 may be operated in the condition known as theoverlap. Operation of the analyzer 1 in overlap condition means that thelong column 7 eluting operation is begun before the eluting operation iscompleted on the short column 6. This may be accomplished due to thefact that an initial period of time elapses during the eluting operationon the column 7 before any of the amino acids of interest are recovered.In this manner, upon completion of the timing cycle 122, an impulse issent from terminal 107 through the switch 97 to begin the operation ofthe long column analysis rather than relying on an impulse to be sentfrom the short column timer 12a. in this manner it is possible for thelong column analysis to begin while the short column analysis is stillbeing carried out thereby providing optimum operating effciency. Itshould be understood that the timing for the eluting step on the shortcolumn and the long column are so arranged that the long column cyclemay be begun prior to the termination of the short column without lossof any of the amino acids of interest being eluted.

From the foregoing, it is now apparent that a novel automatic amino acidanalyzer meeting the objects and advantages set out hereinbefore, aswell as other objects and advantages apparent in the disclosure, isprovided and that changes or modifications as to the preciseconfigurations, shapes and details of the construction set forth in thedisclosure and drawings by way of illustration may be made by thoseskilled in the art without departing from the spirit of the invention,as defined by the claims which follow.

lclaim:

1. Apparatus for determining the quantity of amino acids present in ahydrolized sample which is deposited on and eluted from an ion exchangecolumn comprising a frame, an ion exchange column on said frame, asolenoid operated control valve for controlling the supply of sample andbuffer reagents to said column, said control valve having sampleretention means connected thereto of a predetermined size for retaininga predetermined quantity of sample, said control valve being movablebetween a deactivated position permitting pressure fluid flow of thebuffer reagents through said sample retention means to the ion exchangecolumn for effecting the eluting of the sample and an activated positionpermitting pressure fluid flow of a sample into said sample retentionmeans to obtain the predetermine quantity of said sample while saidcontrol valve maintains pressure fluid flow of the buffer reagentsdirectly with the ion exchange column, means on said frame fordetermining and recording the quantity of amino acids present in thesample as eluted from said column by the buffer reagents and automaticcircuitry means on said frame for controlling said solenoid operatedcontrol valve and said determining and recording means to perform thesteps of the analysis cycle at predetermined times, said automaticcircuitry means including a portion responsive to the completion of ananalysis cycle to move said solenoid operated valve from the deactivatedto the activated position and to supply another sample to said sampleretention means on said control valve and start another analysis cycle.

2. The apparatus according to claim 1 wherein the sample supply meanscomprises a turntable adapted to receive a plurality of samples, adipper mechanism insertable into successive ones of the samples, saiddipper mechanism being in fluid flow connection with said sampleretention means for supplying sample thereto, said automatic circuitrymeans being ef fective in timed sequence to perform the analysis cycleincluding the steps of energizing said turntable to present a sample tosaid dipper mechanism, activating said dipper mechanism to supply asample to said control valve, activating said control valve to supplythe sample to the sample retention means, and deactivating said controlvalve permitting the eluting of the sample to be completed 3. Apparatusfor determining the quantity of amino acids present in a hydrolizedsample comprising a frame, a short ion exchange column on said frame, along ion exchange column on said frame, a pair of solenoid operatedcontrol valves having sample retention means of a predetermine sizeconnected thereto for retaining a predetermined quantity of a sample,said control valves controlling the supply of sample and buffer reagentsto said long and short columns, respectively, said control valves beingmovable between a deactivated position permitting pressure fluid flow ofthe buffer reagents through said sample retention means to the ionexchange columns for effecting the sorbing of the sample onto the ionexchange column and the eluting of the sample therefrom and an activatedposition permitting pressure fluid flow of a sample into said sampleretention means while establishing pressure fluid flow of the bufferreagents directly with the ion exchange columns, transmitting means onsaid frame responsive to the effluent of the eluting operation from saidlong and short columns for transmitting signals to indicate the quantityof amino acids present in the sample, automatic circuitry means on saidframe for controlling the analysis cycle, said automatic circuitry meansbeing effective to control the eluting operation on said long and shortcolumns and including a portion thereof for supplying an impulse to urgesaid solenoid valves from the deactivated to the activated positions,said automatic circuitry means being effective to deactivate the shortcolumn solenoid operated control valve for sorbing and eluting thesample on said short column, upon completion of the short column elutingoperation said automatic circuitry means being effective to deactivatethe long column solenoid operated control valve for sorbing and elutingthe sample on said long column and another portion of said automaticcircuitry means being responsive to the completion of the analysis cycleto start another analysis cycle.

4. The apparatus according to claim 3 including sample detection meansin said automatic circuitry means for turning off said automaticcircuitry mean in the absence of a sample.

5. The apparatus according to claim 3 wherein the sample supply meanscomprises a turntable adapted to receive a plurality of samples, adipper mechanism insertable into successive ones of the samples, saiddipper mechanism being in fluid flow connection with said long and shortcolumn control valves for supplying sample thereto, said turntable beingmovable in response to an impulse from said automatic cir cuitry meansto present a sample beneath said dipper mechanism, said dipper mechanismbeing inserted into a sample for a predetermined time in response to asignal from said sample retention means of the automatic circuitry meansto supply a sample to said long and short column control valves, andsample detection means engageable with the sample to be presented to thedipper mechanism, said sample detection means in the absence of a samplebeing effective to turn off said automatic circuitry means.

6. The apparatus according to claim 5 including a refrigeration unitconnected to said frame, the turntable of samples being maintained insaid refrigeration unit to permit the storage of a plurality of samplessubstantially without deteri oration of any of the amino acids presentin the samples.

7. The apparatus according to claim 5 including a pump means connectedto said dipper mechanism for effecting fluid flow of the samples to saidlong and short column control valves, said pump means being responsiveto an impulse from said automatic circuitry means to initially flush thesample supply system by passing air therethrough and thereafter saiddipper mechanism is inserted into the sample to supply the sample tosaid long and short column control valves.

8. The apparatus according to claim 3 wherein said automatic circuitrymeans includes a central timer adapted to control the analysis of asample in a predetermined timed sequence, a short column timer connectedto said central timer for controlling the amino acid analysis to becarried out on the short column, and a plurality of other timersconnected to said central timer for controlling the amino acid analysesto be carried out on the long column, said short column timer and saidother timers being energized in response to impulses from said centraltimer to effect the complete analysis of a sample, and one of said othertimers controlling the long column analysis being effective to transmitan impulse to said central timer upon completion of the analysis cycleto begin an analysis cycle on another sample.

9. The apparatus according to claim 8 wherein said one timer iseffective to time the complete eluting operation on said long columnthereby transmitting an impulse to said central timer as theregeneration of said long column is completed.

10. The apparatus according to claim 8 wherein upon completion of theanalysis on said short column the short column timer transmits animpulse to the central timer to energize said other timers.

11. The apparatus according to claim 10 including a switch therein, saidswitch being movable between a first position connecting said shortcolumn timer with said central timer and a second position connecting asecond of said other timers with said central timer, the second of saidother timers being effective to time the complete eluting andregeneration operation on said long column, when said switch is in thesecond position the analysis will be operated in overlap condition andupon completion of the complete eluting and regeneration operation onsaid long column an impulse from the second of said other timers will betransmitted to said central timer to begin an analysis cycle on anothersample.

12. Apparatus for determining the quantity of amino acids present in ahydrolyzed sample comprising a frame, short and long ion exchangecolumns on said frame, a short column buffer reagent supply, a longcolumn buffer reagent supply, a control valve connected to said longcolumn and controlling the long column buffer reagents supplied thereto,another control valve connected to said short column and controlling theshort column buffer supplied thereto, sample supply means connected tosaid long and short column control valves, said long and short columncontrol valves each including a sample retention means of apredetermined size for retaining a predetermined quantity of sample andcontrolling the supply of sample to said long and short columns, saidcontrol valves being movable between a deactivated position permittingpressure fluid flow of the buffer reagents through said sample retentionmeans to the respective ion exchange column for effecting the eluting ofthe sample and an activated position permitting pressure fluid flow of asample into said sample retention means to obtain the predeterminequantity of said sample while said control valve maintains pressurefluid flow of the buffer reagents directly with the respective ionexchange column, said long and short columns being connected with theremainder of the eluting system including a ninhydrin system andcolorimeter, said colorimeter being effective to determine theconcentration of amino acids present in the effluent from the elutingoperation of said long and short columns to send signals to a recordermechanism and record same, automatic circuitry means for sequentiallyeffecting the complete analysis cycle and including timer means for theshort column and timer means for the long columns, said automaticcircuitry means activating said sample supply means to supply a sampleto the sample retention means of said long and short column controlvalves, said automatic circuitry means thereafter permitting said shortcolumn control valve to connect the sample in the sample retention meansand said short column buffer reagent with said short column, whilesubstantially simultaneously energizing the short column timer means tocontrol the eluting system for said short column, upon completion of theeluting operation and deenergization of the short column timer means theautomatic circuitry means permitting said long column control valve toconnect the sample retention means and said long column buffer reagentswith said long column while substantially simultaneously energizing thelong column timer means to control the eluting system for the longcolumn, and upon completion of the eluting operation and deenergizing ofsaid long column timer means an impulse is sent to said automaticcircuitry means serving to present another sample to said sample supplymeans and begin another analysis cycle

2. The apparatus according to claim 1 wherein the sample supply meanscomprises a turntable adapted to receive a plurality of samples, adipper mechanism insertable into successive ones of the samples, saiddipper mechanism being in fluid flow connection with said sampleretention means for supplying sample thereto, said automatic circuitrymeans being effective in timed sequence to perform the analysis cycleincluding the steps of energizing said turntable to present a sample tosaid dipper mechanism, activating said dipper mechanism to supply asample to said control valve, activating said control valve to supplythe sample to the sample retention means, and deactivating said controlvalve permitting the eluting of the sample to be completed.
 3. Apparatusfor determining the quantity of amino acids present in a hydrolizedsample comprising a frame, a short ion exchange column on said frame, along ion exchange column on said frame, a pair of solenoid operatedcontrol valves having sample retention means of a predetermine sizeconnected thereto for retaining a predetermined quantity of a sample,said control valves controlling the supply of sample and buffer reagentsto said long and short columns, respectively, said control valves beingmOvable between a deactivated position permitting pressure fluid flow ofthe buffer reagents through said sample retention means to the ionexchange columns for effecting the sorbing of the sample onto the ionexchange column and the eluting of the sample therefrom and an activatedposition permitting pressure fluid flow of a sample into said sampleretention means while establishing pressure fluid flow of the bufferreagents directly with the ion exchange columns, transmitting means onsaid frame responsive to the effluent of the eluting operation from saidlong and short columns for transmitting signals to indicate the quantityof amino acids present in the sample, automatic circuitry means on saidframe for controlling the analysis cycle, said automatic circuitry meansbeing effective to control the eluting operation on said long and shortcolumns and including a portion thereof for supplying an impulse to urgesaid solenoid valves from the deactivated to the activated positions,said automatic circuitry means being effective to deactivate the shortcolumn solenoid operated control valve for sorbing and eluting thesample on said short column, upon completion of the short column elutingoperation said automatic circuitry means being effective to deactivatethe long column solenoid operated control valve for sorbing and elutingthe sample on said long column and another portion of said automaticcircuitry means being responsive to the completion of the analysis cycleto start another analysis cycle.
 4. The apparatus according to claim 3including sample detection means in said automatic circuitry means forturning off said automatic circuitry means in the absence of a sample.5. The apparatus according to claim 3 wherein the sample supply meanscomprises a turntable adapted to receive a plurality of samples, adipper mechanism insertable into successive ones of the samples, saiddipper mechanism being in fluid flow connection with said long and shortcolumn control valves for supplying sample thereto, said turntable beingmovable in response to an impulse from said automatic circuitry means topresent a sample beneath said dipper mechanism, said dipper mechanismbeing inserted into a sample for a predetermined time in response to asignal from said sample retention means of the automatic circuitry meansto supply a sample to said long and short column control valves, andsample detection means engageable with the sample to be presented to thedipper mechanism, said sample detection means in the absence of a samplebeing effective to turn off said automatic circuitry means.
 6. Theapparatus according to claim 5 including a refrigeration unit connectedto said frame, the turntable of samples being maintained in saidrefrigeration unit to permit the storage of a plurality of samplessubstantially without deterioration of any of the amino acids present inthe samples.
 7. The apparatus according to claim 5 including a pumpmeans connected to said dipper mechanism for effecting fluid flow of thesamples to said long and short column control valves, said pump meansbeing responsive to an impulse from said automatic circuitry means toinitially flush the sample supply system by passing air therethrough andthereafter said dipper mechanism is inserted into the sample to supplythe sample to said long and short column control valves.
 8. Theapparatus according to claim 3 wherein said automatic circuitry meansincludes a central timer adapted to control the analysis of a sample ina predetermined timed sequence, a short column timer connected to saidcentral timer for controlling the amino acid analysis to be carried outon the short column, and a plurality of other timers connected to saidcentral timer for controlling the amino acid analyses to be carried outon the long column, said short column timer and said other timers beingenergized in response to impulses from said central timer to effect thecomplete analysis of a sample, and one of said other timers cOntrollingthe long column analysis being effective to transmit an impulse to saidcentral timer upon completion of the analysis cycle to begin an analysiscycle on another sample.
 9. The apparatus according to claim 8 whereinsaid one timer is effective to time the complete eluting operation onsaid long column thereby transmitting an impulse to said central timeras the regeneration of said long column is completed.
 10. The apparatusaccording to claim 8 wherein upon completion of the analysis on saidshort column the short column timer transmits an impulse to the centraltimer to energize said other timers.
 11. The apparatus according toclaim 10 including a switch therein, said switch being movable between afirst position connecting said short column timer with said centraltimer and a second position connecting a second of said other timerswith said central timer, the second of said other timers being effectiveto time the complete eluting and regeneration operation on said longcolumn, when said switch is in the second position the analysis will beoperated in overlap condition and upon completion of the completeeluting and regeneration operation on said long column an impulse fromthe second of said other timers will be transmitted to said centraltimer to begin an analysis cycle on another sample.
 12. Apparatus fordetermining the quantity of amino acids present in a hydrolyzed samplecomprising a frame, short and long ion exchange columns on said frame, ashort column buffer reagent supply, a long column buffer reagent supply,a control valve connected to said long column and controlling the longcolumn buffer reagents supplied thereto, another control valve connectedto said short column and controlling the short column buffer suppliedthereto, sample supply means connected to said long and short columncontrol valves, said long and short column control valves each includinga sample retention means of a predetermined size for retaining apredetermined quantity of sample and controlling the supply of sample tosaid long and short columns, said control valves being movable between adeactivated position permitting pressure fluid flow of the bufferreagents through said sample retention means to the respective ionexchange column for effecting the eluting of the sample and an activatedposition permitting pressure fluid flow of a sample into said sampleretention means to obtain the predetermine quantity of said sample whilesaid control valve maintains pressure fluid flow of the buffer reagentsdirectly with the respective ion exchange column, said long and shortcolumns being connected with the remainder of the eluting systemincluding a ninhydrin system and colorimeter, said colorimeter beingeffective to determine the concentration of amino acids present in theeffluent from the eluting operation of said long and short columns tosend signals to a recorder mechanism and record same, automaticcircuitry means for sequentially effecting the complete analysis cycleand including timer means for the short column and timer means for thelong columns, said automatic circuitry means activating said samplesupply means to supply a sample to the sample retention means of saidlong and short column control valves, said automatic circuitry meansthereafter permitting said short column control valve to connect thesample in the sample retention means and said short column bufferreagent with said short column, while substantially simultaneouslyenergizing the short column timer means to control the eluting systemfor said short column, upon completion of the eluting operation anddeenergization of the short column timer means the automatic circuitrymeans permitting said long column control valve to connect the sampleretention means and said long column buffer reagents with said longcolumn while substantially simultaneously energizing the long columntimer means to control the eluting system for the long column, and uponcompletion of the eluting operation aNd deenergizing of said long columntimer means an impulse is sent to said automatic circuitry means servingto present another sample to said sample supply means and begin anotheranalysis cycle.